The beta-globin genes were among the first human genes to be cloned. This led to expectations that hemoglobinopathy diseases such as sickle cell anemia and beta-thalassemia would be among the first diseases to be treated by gene therapy. And yet, nearly ten years after the first gene therapy protocol was approved and after a total of 200 therapeutic protocols have been submitted, no hemoglobinopathy patient has been treated with gene replacement therapy. This is largely due to technical problems involving the gene therapy vectors to be used for the hemoglobinopathies. One of these problems is that consistent, long-lasting, high-level expression has yet to be attained in pre-clinical systems. Evidence indicates that this problem is, at least in part, due to an inability of current vectors to efficiently create a transcriptionally active or "open" chromatin structure surrounding the integrated vector. The normal beta-globin genes, in the context of the beta-globin gene locus, are able to efficiently perform this process in erythroid cells. Our long term goals are to study the processes by which the normal locus is able to open chromatin structure in an erythroid- specific fashion and to then apply these findings to help develop new gene therapy vectors which are able to independently open chromatin structure wherever they insert into the human genome. We are attacking these problems on four fronts. First we will continue to characterize a 101bp element from HS4 of the beta-globin LCR which is able to locally open chromatin structure in erythroid cells. Our current data implicates the transcription factors NF-E2, GATA-1 and Sp1 in this process. GATA-1 seems to be particularly important in this process. In our second aim we will perform structural studies on this factor gain insight the mechanisms by which it is able to reorganize chromatin structure. In our third aim we will continue our efforts to locate the boundaries of the domain of open chromatin structure which forms around the globin gene locus. Finally, we will continue to test the ability of chromatin structure-determining elements (i.e., HS4 HSFE, chicken beta-globin boundary element, HS3 core, HS2 enhancer) alone and in combination to yield position-independent, high-level, non-silencing expression. These experiments provide a comprehensive approach to understanding the regulation of beta-globin locus chromatin structure and potential applications to development of gene therapy strategies.